Over the last four decades, antibodies have enabled extraordinary advances in biomedical research and have demonstrated considerable efficacy in the clinic as therapeutic agents. Despite these remarkable successes, the biophysical properties of antibodies limit their therapeutic potential and further development as high- performance affinity reagents. Due to the presence of stabilizing disulfide bridges within the antibody architecture, it is exceedingly difficult to isolate functional antibody fragments within the reducng environment of the cytoplasm. As a result, most intracellularly-expressed antibodies are folded poorly in vivo and aggregate, ultimately resulting in nonfunctional affinity reagents that are rapidly degraded. Importantly, this same issue prevents routine antibody expression via high throughput in vitro transcription and translation (IVTT) methodologies, as these protocols require reducing agents to preserve protein synthesis activity. I propose a systematic protein engineering and selection approach to generate antibodies amenable to intracellular expression and IVTT. This is based on the hypothesis that genetically fusing heterodimerizing leucine zipper domains onto antibody fragments will stabilize the molecules enough to permit the removal of the disulfide-forming cysteines. Then, optimization of their frameworks through directed evolution should produce stable antibodies that are resistant to reducing agents and are therefore amenable to both intracellular expression as well as IVTT. I will begin this process by replacing the interchain cysteines on antibody fragments (Fabs) with hydrophobic residues using site-directed mutagenesis. Subsequently, I will genetically fuse non-human leucine zippers onto the carboxy-terminal ends of the Fab scaffolds. Evaluation of these engineering procedures will be performed using SDS-PAGE gel and several thermostability assays. I will go on to sequentially replace the two cysteines in each domain of the Fab with hydrophobic residues to convert the scaffold to a disulfide-free format. After every round of mutation, I will use ribosome display to enrich for the most stable mutants and use these as templates in the next round. This affinity maturation process will be performed iteratively until Fabs are obtained with sufficient stability and affinity for our purposes. These Fabs will be functionally tested for intracellular expression in mammalian cells, and will then be adapted and optimized for high-throughput manufacture in 96-well format via in vitro transcription and translation protocols. Success of this project would enable unprecedented studies of protein function and would accelerate research in all fields related to the life sciences. Moreover, as part of a larger collaborative effort with the Recombinant Antibody Network (RAN), these studies will help establish a robust and efficient pipeline for the generation of a renewable, validated, and standardized set of antibody reagents freely distributed to academic researchers and to the general scientific community alike. PUBLIC HEALTH RELEVANCE: The enormous potential of recombinant antibodies as intracellular immunotherapeutics has been hampered by the difficulty in isolating functional antibody fragments within the reducing environment of the cytoplasm where stabilizing disulfide bonds cannot form. Importantly, this same issue has limited the routine production of these affinity reagents to low-throughput methodologies. This proposal focuses on genetically engineering a highly stable, disulfide-free recombinant antibody scaffold resistant to reducing agents making it amenable to intracellular expression and high-throughput synthesis via in vitro transcription and translation technologies.